At present, a large number of microorganisms are known to store polyester as an energy source substance within cells. A typical example of the polyester is poly-3-hydroxybutyric acid (hereinafter referred to briefly as P(3HB)), which is a homopolymer of 3-hydroxybutyric acid (hereinafter referred to as 3HB for short). It was first discovered in Bacillus megaterium (M. Lemoigne, Ann. Inst. Pasteur, 39, 144 (1925)). P(3BH) is a thermoplastic polymer and is biodegradable in the natural environment and, thus, has recently attracted attention as an ecofriendly plastic. However, P(3HB) is high in crystallinity, and hard and fragile by nature, so that the range of practical application thereof is limited. Therefore, studies have been undertaken to modify the same for bringing about improvements in these properties.
In Japanese Kokai Publication Sho-57-150393 and Japanese Kokai Publication Sho-59-220192, among others, a technology of producing a copolymer made of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) (hereinafter such copolymer is referred to as P(3HB-co-3HV)) is disclosed. This P(3HB-co-3HV) is rich in flexibility as compared with P(3HB), hence was considered to have a wide application range. In actuality, however, P(3HB-co-3HV) shows only slight changes in physical properties even when the mole fraction of 3HV is increased. In particular, the flexibility, which is required for its use in films and the like, will not be improved. Thus, it has been used only in the field of rigid shaped articles such as shampoo bottles and disposable razor grips.
In recent years, studies have been made concerning copolyesters consisting of two components 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as 3HH for short) (hereinafter such copolyesters are referred to as P(3HB-co-3HH) for short), as described in Japanese Kokai Publication Hei-05-93049 and Japanese Kokai Publication Hei-07-265065, among others. According to these publications, this technology of producing P(3HB-co-3HH) comprises fermentative production thereof from a fatty acid, such as oleic acid, or an oil or fat, such as olive oil, using Aeromonas caviae isolated from soil. Studies concerning the properties of P(3HB-co-3HH) have also been made (Y. Doi, S. Kitamura, H. Abe, Macromolecules, 28, 4822-4823 (1995)). According to this report, when A. caviae is cultured using a fatty acid containing not less than 12 carbon atoms as the only carbon source, P(3HB-co-3HH) with a 3HH content of 11 to 19 mole percent can be fermentatively produced. It has been revealed that the properties of such P(3HB-co-3HH) change from hard and brittle gradually to soft and flexible, to an extent exceeding the flexibility of P(3HB-co-3HV), with the increase in mole fraction of 3HH. However, the above method of production is low in productivity, namely the yield of cells is 4 g/L and the polymer content is 30%. Therefore, methods capable of attaining higher productivity for practical use have been searched for.
A PHA (polyhydroxyalkanoic acid) synthase gene has been cloned from Aeromonas caviae, which is a producer of P(3HB-co-3HH) (T. Fukui, Y. Doi, J. Bacteriol., vol. 179, No. 15, 4821-4830 (1997); Japanese Kokai Publication Hei-10-108682). This gene was transformed into Ralstonia eutropha (formerly Alcaligenes eutrophus), and cultivation was carried out using the resulting transformant and a vegetable oil as the carbon source, whereby a content in cells of 4 g/L and a polymer content of 80% were attained (T. Hukui et al., Appl. Microbiol. Biotechnol., 49, 333 (1998)). A method of producing P(3HB-co-3HH) using a bacterial species, such as Escherichia coli, or a plant as the host has also been disclosed (WO 00/43525), without describing any productivity data, however.
The above-mentioned polymer P(3HB-co-3HH) can be given a wide range of physical properties, from properties of rigid polymers to properties of flexible polymers, by changing the mole fraction of 3HH and therefore can be expected to be applicable in a wide range, from television boxes and the like, for which rigidity is required, to yarns, films and the like, for which flexibility is required. However, the production methods mentioned above are still poor in the productivity of P(3HB-co-3HH). There is no other way but to say that they are still unsatisfactory as production methods for the practical use of P(3HB-co-3HH).
In a recent study of the production of biodegradable polyesters, Leaf et al. used yeast high in cell productivity as the host (Microbiology, vol. 142, pp. 1169-1180 (1996)). Thus, the polyester synthase gene of Ralstonia eutropha was transformed into Saccharomyces cerevisiae, a kind of yeast, the thus-produced transformant was cultured using glucose as the carbon source, and the accumulation of P(3HB) was confirmed (polymer content 0.5%). However, the polymer produced in this study was that hard and brittle P(3HB).
Yeast is known to grow fast and be high in cell productivity. Among them, yeasts belonging to the genus Candida attracted attention as single cell proteins in the past and, since then, studies have been made on the production of cells thereof for use as feeds using normal-paraffins as carbon sources. Further, in recent years, vectors functioning in hosts belonging to the genus Candida have been developed, and the production of substances using the recombinant DNA technology has been reported (Kagaku to Seibutsu, vol. 38, No. 9, 614 (2000)). When Candida utilis is used as the host, the α-amylase productivity is as high as about 12.3 g/L. Microorganisms of the genus Candida having such high substance productivity are expected to serves as hosts in polymer production. Furthermore, cells thereof can be separated from the culture fluid with ease as compared with bacteria and, thus, the polymer extraction and purification steps can be facilitated.
Thus, the advent of a method of producing P(3HB-co-3HH) having good physical properties using yeast belonging to the genus Candida has been waited for.